The present invention relates to substrates and methods for determining enzymes. More particularly, the invention relates to qualitative and quantitative methods for determining proteolytic enzymes.
The determination of specific enzymes in biological fluids, such as blood, tissue homogenates, and cytoplasm can be very useful for the diagnosis of certain diseases. The discovery of synthetic substrates for such determinations has resulted in clinical assay procedures having a high degree of specificity, reliability, and sensitivity. Such substrates have been employed for the determination of amylase (Driscoll, R. C., et al., U.S. Pat. No. 4,102,747) and various proteinases.
Synthetic proteinase substrates have generally been amino acid derivatives of aromatic amines. The number and arrangement of amino acids in the peptide moiety determine the enzyme specificity of the substrate and the enzyme activity is measured by the amount of aromatic amine moiety liberated upon hydrolysis of the substrate. Amino acid derivatives of p-nitroaniline have been widely used as synthetic substrates. Erlanger, B. F., U.S. Pat. No. 3,412,150. Other aromatic amines which have been reacted with amino acids or peptides include 2-naphthylamine, 4-methoxy-2-naphthylamine, and 7-amino-4-methylcoumarin. The use of 2-naphthylamine and 4-methoxy-2-naphthylamine for the preparation of synthetic substrates and prior art relating thereto are discussed by Smith, R. E., U.S. Pat. No. 3,862,011. Peptide derivatives of 7-amino-4-methylcoumarin have recently been reported as fluorogenic substrates for a number of proteinases. Zimmerman, M., Yurewicz, E., Patel, G., Anal. Biochem. 70, 258-262 (1976) and Zimmerman, M., Quigley, J. P., Ashe, B., Dorn, C., Goldfarb, R., Troll, W., Proc. Natl. Acad. Sci., 75, 750-753 (1978).
Because the chromophore, p-nitroaniline, is yellow, enzyme assays employing that chromophore are colorimetric. Fluorescence assays are sometimes preferred over colorimetric assays, because of greater sensitivity and less background interference. The aromatic amine chromophores heretofore used to prepare synthetic substrates are fluorescent, but their fluorescence generally occurs in the blue region of the spectrum. Such fluorescence is disadvantageous, because it is difficult to measure with inexpensive instruments, and it is similar to fluorescense of other materials present in the analyte, including, in some instances, the intact substrate. These assays are useful for cytological studies for the detection of an enzyme within a single cell. When such cells are viewed under a fluorescence microscope, a blue color is difficult to see or distinguish from the background, but cells emitting light in the yellow region of the spectrum are easily visualized.
To overcome these problems, investigators have focused on reactions involving the enzyme-liberated chromophore to enhance color or fluorescence at a desired wavelength. For instance, aromatic amine chromophores may be reacted with diazonium salts to form azo dyes which are determined spectrophotometrically. In U.S. patent application Ser. No. 828,394, R. E. Smith, et al. disclose a reaction of the aromatic amine chromophore with certain aromatic aldehydes to form Shiff base compounds which fluoresce in the yellow-green region of the spectrum.
Although such methods have each constituted significant advances over the prior art, there is a need for synthetic substrates for proteinase enzymes which do not fluoresce in the yellow region, but which upon enzyme hydrolysis, release a chromophore which fluoresces strongly in that region of the spectrum. Such substrates would, thus, obviate the need for further reactions involving the liberated chromophore, and the concentration of such chromophore could be readily determined by a fluorometric technique.